Compositions containing, methods involving, and uses of non-natural amino acid linked dolastatin derivatives

Abstract

Disclosed herein are non-natural amino acids and dolastatin analogs that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The dolastatin analogs can include a wide range of possible functionalities, but typically have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid dolastatin analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Typically, the modified dolastatin analogs include at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid dolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use.

1 .- 24 . (canceled) 
     
     
         25 . A compound comprising Formula (VIII) or (IX), wherein the compound is a trastuzumab antibody conjugated to a dolastatin, wherein the conjugation occurs via a non-naturally encoded amino acid in the antibody, wherein Formula (VIII) or (IX) correspond to: 
       
         
           
           
               
               
           
         
         wherein:
 A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; 
 B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — where k is 1, 2, or 3, —S(O) k -(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O) k N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′) 2 —N═N—, and —C(R′) 2 —N(R′)—N(R′)—,
 wherein each R′ is independently H, alkyl, or substituted alkyl; 
 
 R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; 
 R 1  is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; 
 R 2  is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide;
 wherein at least one of R 1  and R 2  is a polypeptide, wherein the polypeptide is the trastuzumab antibody, wherein amino acid position 121 of the antibody is substituted with the non-naturally encoded amino acid; 
 
 R 3  and R 4  are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3  and R 4  or two R 3  groups optionally form a cycloalkyl or a heterocycloalkyl; 
 Z has the structure of: 
 
       
       
         
           
           
               
               
           
         
         
           
             R 5  is H, COR 8 , C 1 -C 6 alkyl, or thiazole;
 R 8  is OH; 
 
             R 6  is OH or H; 
             Ar is phenyl or pyridine; 
             R 7  is C 1 -C 6  alkyl or hydrogen; and 
           
           L is a linker selected from the group consisting of -alkylene-, -alkylene-C(O)—, -(alkylene-O) n -alkylene-, -(alkylene-O) n -alkylene-C(O)—, -(alkylene-O) n —(CH 2 ) n′ —NHC(O)—(CH 2 ) n″ —C(Me) 2 -S—S—(CH 2 ) n′″ —NHC(O)-(alkylene-O) n″″ -alkylene-, -(alkylene-O) n -alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O) n -alkylene-U-alkylene-C(O)—, and -(alkylene-O) n -alkylene-U-alkylene-; wherein:
 W has the structure of: 
 
         
       
       
         
           
           
               
               
           
         
         
           
             U has the structure of: 
           
         
       
       
         
           
           
               
               
           
         
         
           
             and 
             each n, n′, n″, n′″ is independently an integer from 0 to 20; 
           
           wherein substituted means substituted with one or more substituents independently selected from the group consisting of halo, C 1 -C 10  alkyl, C 2 -C 10  alkenyl, C 2 -C 10  alkynyl, C 1 -C 10  alkoxy, C 5 -C 12  aralkyl, C 3 -C 12  cycloalkyl, C 4 -C 12  cycloalkenyl, phenyl, toluolyl, xylenyl, biphenyl, C 2 -C 12  alkoxyalkyl, C 5 -C 12  alkoxyaryl, C 5 -C 12  aryloxyalkyl, C 2 -C 12  oxyaryl, C 1 -C 6  alkylsulfonyl, C 1 -C 10  alkylsulfonyl, —(CH 2 ) m —O—(C 1 -C 10  alkyl), aryl, fluoroalkyl, heterocyclic radical, nitroalkyl, —NO 2 , —CN, —NR″C(O)—(C 1 -C 10  alkyl), —C(O)—(C 1 -C 10  alkyl), C 2 -C 10  alkthioalkyl, —C(O)O—(C 1 -C 10  alkyl), —OH, —SO 2 , ═S, —COOH, —NR″ 2 , carbonyl, —C(O)—(C 1 -C 10  alkyl)—CF 3 , —C(O)—CF 3 , —C(O)NR″ 2 , —(C 1 -C 10  aryl)-S—(C 6 -C 10  aryl), —C(O)—(C 6 -C 0  aryl), —(CH 2 ) m —O—(CH 2 ) m —O—(C 1 -C 10  alkyl), —C(O)NR″, —C(S)NR″ 2 , —SO 2 NR″ 2 , —NR″C(O)NR″ 2  and —NR″C(S)NR″ 2 ; wherein each R″ group is independently H, alkyl, aryl or alkaryl; and each m is from 1 to 8; 
         
         or a pharmaceutically acceptable salt or solvate thereof. 
       
     
     
         26 .- 57 . (canceled) 
     
     
         58 . The compound of  claim 25 , wherein the conjugation occurs via the non-naturally encoded amino acid at amino acid position 121 of the antibody. 
     
     
         59 . The compound of  claim 58 , wherein:
 A is arylene;   B is absent;   Ar is phenyl;   L is -(alkylene-O) n -alkylene-;   R is alkyl;   each of R 1  and R 2  is a polypeptide, wherein the polypeptide is the trastuzumab antibody;   R 3  and R 4  are H;   R 5  is COR 8 ;   R 6  is H; and   R 7  is C 1 -C 6  alkyl.   
     
     
         60 . The compound of  claim 59 , wherein the compound is a compound of Formula (VIII). 
     
     
         61 . The compound of  claim 59 , wherein the compound is a compound of Formula (IX). 
     
     
         62 . A pharmaceutical composition comprising a compound of  claim 59  and a pharmaceutically acceptable carrier, excipient or binder. 
     
     
         63 . A method for treating a solid tumor overexpressing HER2 in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a compound of  claim 59 . 
     
     
         64 . The method of  claim 63 , wherein the solid tumor is breast cancer, small cell lung carcinoma, ovarian cancer, prostate cancer, gastric carcinoma, cervical cancer, esophageal carcinoma or colon cancer. 
     
     
         65 . The method of  claim 64 , wherein the solid tumor is breast cancer. 
     
     
         66 . The method of  claim 65 , wherein the compound is a compound of Formula (VIII).